Mammalian Cell Culture Media Research Articles

The addition of mammalian cell culture medium impacts nanoparticle toxicity in zebrafish

Engineered nanomaterials (ENMs) are ubiquitous in contemporary applications, yet their environmental and human health impacts remain inadequately understood. This study addresses the challenge of identifying potential risks associated with ENM exposure by highlighting the significant variability in existing research methodologies. Without a systematic collection of toxicological data that encompasses standardized materials, relevant platforms, and assays, the task of identifying potential risks linked to ENM exposure becomes an intricate challenge. In vitro assessments often use media rich in ionic species, such as RPMI and fetal bovine serum (FBS). Zebrafish embryos, known to develop normally in low-ionic environments, were exposed to Cerium Oxide, Zinc Oxide, and Graphene Oxides in different media at varying concentrations. Here, we discovered that zebrafish embryos tolerated a mix of 80 % RPMI, 2 % FBS, and 1 % antibiotic cocktail. The results revealed that adverse effects observed in zebrafish with certain nanomaterials in Ultra-Pure (UP) water were mitigated in cell culture medium, emphasizing the importance of revisiting previously considered non-toxic materials in vitro. The zebrafish results underscore the importance of utilizing a multidimensional in vivo platform to gauge the biological activity of nanomaterials accurately.

Антикоррозионные гидроксиапатитсодержащие покрытия для функционализации биорезорбируемых магниевых сплавов

The biocompatible protective coating was formed using the plasma electrolytic oxidation (PEO) on a bioresorbable Mg–0.8Ca alloy. The electrochemical properties and the mechanism of material bioresorption were established. The model of the biodegradation process of the alloy with a hydroxyapatite-containing PEO-coating in mammalian cell culture medium was proposed.

Hydroxyapatite-containing PEO-coating design for biodegradable Mg-0.8Ca alloy: Formation and corrosion behaviour

In this study, the biocompatible protective coating was formed using plasma electrolytic oxidation (PEO) on bioresorbable Mg-0.8Ca alloy. The composition of the formed coating was studied using XRD, SEM-EDX analysis, and micro-Raman spectroscopy. The uniform distribution of hydroxyapatite over the thickness of protective PEO-layer was established. Using traditional (EIS, PDP, OCP) and local scanning electrochemical methods (SVET, SIET with H+-selective microelectrode), the level of protective properties of PEO-layer in a biological environment (mammalian cell culture medium, MEM) was determined. It was established that modification of Mg-0.8Ca alloy surface by PEO contributes to a significant increase in the corrosion resistance of the surface layer, making it possible to control the process of material`s biodegradation. The maximum local electrochemical activity was recorded after 72 h of testing, while for the uncoated sample, intense corrosion degradation was recorded in the first 12 min of exposure to the cell culture medium. Formation of the PEO-coating results in a twofold decrease in the corrosion current density (2.8ˑ10−6 A cm−2) and an increase in the impedance modulus measured at a low frequency (1.7ˑ104 Ω cm2) in comparison with the uncoated material (9.5ˑ10−6 A cm−2; 8.1ˑ103 Ω cm2). The mechanism of material bioresorption was established and a model for biodegradation process of Mg-0.8Ca alloy with hydroxyapatite-containing PEO-coating in MEM was proposed. Analysis of these results and comparing with others obtained by various scientific groups indicate the prospects for application of biocompatible PEO-coating on Mg-Ca alloy in implant surgery.

Conversion of mammalian cell culture media waste to microbial fermentation feed efficiently supports production of recombinant protein by Escherichia coli.

Deriving new value from waste streams through secondary processes is a central aim of the circular bioeconomy. In this study we investigate whether chemically defined spent media (CDSM) waste from cell culture bioprocess can be recycled and used as a feed in secondary microbial fermentation to produce new recombinant protein products. Our results show that CDSM supplemented with 2% glycerol supported a specific growth rate of E. coli cultures equivalent to that achieved using a nutritionally rich microbiological media (LB). The titre of recombinant protein produced following induction in a 4-hour expression screen was approximately equivalent in the CDSM fed cultures to that of baseline, and this was maintained in a 16-hr preparative fermentation. To understand the protein production achieved in CDSM fed culture we performed a quantitative analysis of proteome changes in the E. coli using mass spectrometry. This analysis revealed significant upregulation of protein synthesis machinery enzymes and significant downregulation of carbohydrate metabolism enzymes. We conclude that spent cell culture media, which represents 100s of millions of litres of waste generated by the bioprocessing industry annually, may be valorized as a feed resource for the production of recombinant proteins in secondary microbial fermentations. Data is available via ProteomeXchange with identifier PXD026884.

The detailed corrosion performance of bioresorbable Mg-0.8Ca alloy in physiological solutions

The electrochemical behaviour of biodegradable magnesium alloy Mg-0.8Ca was evaluated in a mammalian cell culture medium (MEM) and NaCl solutions (0.9 wt.%, 0.3 wt.%) using traditional (EIS, PDP, OCP) and local scanning electrochemical (SVET, SIET with pH-selective microelectrode) methods at the micro- and meso-level. Corrosion rates of samples in two different media were determined using weight loss tests. The influence of testing media components, alloy composition and microstructure on the material's degradation process was determined. The SVET/SIET test parameters were optimized for in vitro investigation of the bioresorbable material surface. The mechanism of the alloy's bioresorption was suggested. The effect of microsized phases on the corrosion behaviour of the alloy was proved using complementary in situ monitoring and SKPFM measurements. The rapid degradation rate of the alloy is related to the presence of local microgalvanic cells formed by cathodic α-magnesium matrix and anodic Mg2Ca phase. The highest corrosion activity was revealed in the first 12 min of sample exposure to MEM, followed by stabilization of corrosion process due to the material's passivation. Using SEM-EDX analysis, micro-Raman spectroscopy and XPS analysis the composition of the corrosion products was determined. Degradation in MEM proceeds with a formation of magnesium-and-carbonate substituted hydroxyapatite-containing film on the sample's surface. The low possibility of application of Mg-0.8Ca alloy without coating protection in implant surgery was highlighted.

Loss of the ClpXP Protease Leads to Decreased Resistance to Cell-Envelope Targeting Antimicrobials in Bacillus anthracis Sterne

The ClpX ATPase is critical for resistance to cell envelope targeting antibiotics in Bacillus anthracis, however, it is unclear whether this is due to its function as an independent chaperone or as part of the ClpXP protease. In this study, we demonstrate that antibiotic resistance is due to formation of the ClpXP protease through construction of a ClpX complementation plasmid that is unable to interact with ClpP. Additionally, we genetically disrupted both clpP genes, clpP1 and clpP2, found in B. anthracis Sterne and find that the loss of either increases susceptibility to cell envelope targeting antimicrobials, although neither has as strong of a phenotype as loss of clpX and neither clpP gene is essential for virulence in a G. mellonella model of infection. Lastly, we looked at changes to cell envelope morphology that could contribute to increased antibiotic sensitivity. We find no difference in cell charge or cell lysis, although we do see increased hydrophobicity in the ΔclpX strain, decreased cellular density and slightly thinner cells walls. We also see significant cell division defects in ΔclpX, although only when cells are grown in the mammalian cell culture medium, RPMI. We conclude that the intrinsic resistance of B. anthracis to cell wall active antimicrobials is dependent on formation of the ClpXP protease and that this could be due, at least in part, to the role of ClpX in regulating cell envelope morphology.

A semi‐empirical mathematical model to specify the pH of bicarbonate‐buffered cell culture medium formulations

AbstractThe carbon dioxide/bicarbonate system is routinely used as a buffer in mammalian cell culture medium. However, due to the continuous degassing of carbon dioxide and its dependence on temperature, it is difficult to achieve the target pH during preparation at ambient temperature and without control of dissolved carbon dioxide, and even more so at cell culture operating conditions. These problems were amplified when preparing multiple (e.g., 20) customized preparations of an in‐sourced proprietary medium during research and process development of mammalian cell culture systems. Thus, a mathematical model was created to specify the amount of acid or base to add during preparation so as to achieve the target pH of each medium at process conditions without having to do titration. The relationship between gaseous carbon dioxide and the dissolved carbon dioxide in the proprietary medium containing unknown species was specified using a modified Henry's law equation (as done in prior work). Further, to allow medium preparation without doing titration, the acid/base properties of the proprietary medium were fitted by a parameter related to its Net Medium Acids during specification of model parameters. A subset of the solutions was prepared and tested in bioreactors with controlled CO2 flow to validate the model. Then, the model was used to assess the equivalence of the pHs of the customized medium formulations despite variations in pCO2 that occurred during incubation and sampling.

In Vitro Stimulation of Whole Milk Specimens: A Field-Friendly Method to Assess Milk Immune Activity.

The immune system of milk protects against infections and guides immune system development. A system-level understanding of milk immune activity is critical for research into infant infectious disease risk and lifelong health. To describe a protocol to characterize immune activity in human milk via in vitro stimulation for use in population-based (rather than clinical) research. This study proceeded in two phases, each with a cross-sectional design. Human milk specimens were incubated for 24 hr at 37 °C in mammalian cell culture medium with stimuli (e.g., Salmonella enterica) in a CO2-enriched environment. Immune responses to stimuli were characterized as the change in cytokine: [stimulated]/[baseline]. Predictors of cytokine responses were evaluated with generalized linear models. Patterns were detectable across mother-child dyads: Interleukin-6 responses to stimuli were generally positively associated with child age and with maternal autoimmune disease. Our method allows characterization of pro-inflammatory milk immune activity in vitro in population-based (rather than clinical) research settings. In vitro activity has a system-level interpretation and is likely to be of broad utility in global health research in settings with high infectious disease risk, where understanding the immune system of milk is critical to understanding maternal and child health.

A Simple Method for In-Depth Proteome Analysis of Mammalian Cell Culture Conditioned Media Containing Fetal Bovine Serum.

A conditioned medium of a cell culture is widely used for various biological applications and frequently analyzed to characterize the functional proteins responsible for observed biological functions. However, a large number of abundant proteins in fetal bovine serum (FBS), usually included in the conditioned medium of a mammalian cell culture medium, hampers in-depth proteomic analysis by liquid chromatography–tandem mass spectrometry (LC-MS/MS). For a deep proteomic analysis of a conditioned medium by LC-MS/MS, we developed a simple albumin depletion approach coupled with data-independent acquisition (DIA)-mode LC-MS/MS for the conditioned medium of mammalian cells in this study. The results showed that this approach enabled the detection of more than 3700 cell-derived proteins in the cell culture supernatant containing FBS. We further demonstrated the potency of this approach by analyzing proteins in the conditioned media of HeLa cells with and without tumor necrosis factor (TNF) stimulation: >40 differentially accumulated proteins, including four cytokines, upon TNF stimulation were identified in the culture media, which were hardly detected by conventional proteome approaches in the literature.

Differential transcription profiling of the phage LUZ19 infection process in different growth media

ABSTRACT RNA sequencing of phage-infected bacterial cultures offers a snapshot of transcriptional events occurring during the infection process, providing insights into the phage transcriptional organization as well as the bacterial response. To better mimic real environmental contexts, we performed RNA-seq of Pseudomonas aeruginosa PAO1 cultures infected with phage LUZ19 in a mammalian cell culture medium to better simulate a phage therapy event and the data were compared to lysogeny broth medium. Regardless of the media, phage LUZ19 induces significant transcriptional changes in the bacterial host over time, particularly during early infection (t = 5 min) and gradually shuts down bacterial transcription. In a common response in both media, 56 P. aeruginosa PAO1 genes are differentially transcribed and clustered into several functional categories such as metabolism, translation and transcription. Our data allowed us to tease apart a medium-specific response during infection from the identified infection-associated responses. This reinforces the concept that phages overtake bacterial transcriptome in a strict manner to gain control of the bacterial machinery and reallocate resources for infection, in this case overcoming the nutritional limitations of the mammalian cell culture medium. From a phage therapy perspective, this study contributes towards a better understanding of phage–host interaction in human physiological conditions and demonstrates the versatility of phage LUZ19 to adapt to different environments.

Коррозионная деградация биорезорбируемого сплава Mg–0,8Ca в среде культивирования клеток млекопитающих

Представлены результаты исследования электрохимической активности биорезорбируемого магниевого сплава Mg–0,8Ca в среде для культивирования клеток млекопитающих – minimum essential medium (MEM). Наиболее высокая коррозионная активность сплава выявлена на начальной стадии выдержки в растворе (первые 12 мин). Установлен механизм коррозионной деградации материала, заключающийся в инициировании разрушения на границах зерен вследствие образования микрогальванических пар. Это объясняется присутствием в сплаве интерметаллидной фазы Mg2Ca, являющейся анодной по отношению к α-матрице магния. Предложена модель формирования слоя продуктов коррозии на поверхности сплава при его выдержке в MEM. The paper presents results of study of electrochemical activity of the Mg–0.8Ca bioresorbable alloy in a medium for the cultivation of mammalian cells – the minimum essential medium (MEM). The highest corrosion activity of the alloy was revealed at the initial stage of exposure in the solution (for the first 12 minutes). The mechanism of corrosion degradation of material has been determined and it is characterized with initiation of the destruction at grain boundaries caused by the formation of micro-galvanic couples. It is explained by the presence of Mg2Ca intermetallic phase which is anodic against α- magnesium matrix. A model of formation of corrosion products layer on alloy`s surface during the exposition in MEM solution is proposed.

From UV to NIR: A Full-Spectrum Metal-Free Photocatalyst for Efficient Polymer Synthesis in Aqueous Conditions.

Photo-mediation offers unparalleled spatiotemporal control over controlled radical polymerizations (CRP). Photo-induced electron/energy transfer reversible addition-fragmentation chain transfer (PET-RAFT) polymerization is particularly versatile owing to its oxygen tolerance and wide range of compatible photocatalysts. In recent years, broadband- and near-infrared (NIR)-mediated polymerizations have been of particular interest owing to their potential for solar-driven chemistry and biomedical applications. In this work, we present the first example of a novel photocatalyst for both full broadband- and NIR-mediated CRP in aqueous conditions. Well-defined polymers were synthesized in water under blue, green, red, and NIR light irradiation. Exploiting the oxygen tolerant and aqueous nature of our system, we also report PET-RAFT polymerization at the microliter scale in a mammalian cell culture medium.

From UV to NIR: A Full‐Spectrum Metal‐Free Photocatalyst for Efficient Polymer Synthesis in Aqueous Conditions

AbstractPhoto‐mediation offers unparalleled spatiotemporal control over controlled radical polymerizations (CRP). Photo‐induced electron/energy transfer reversible addition–fragmentation chain transfer (PET‐RAFT) polymerization is particularly versatile owing to its oxygen tolerance and wide range of compatible photocatalysts. In recent years, broadband‐ and near‐infrared (NIR)‐mediated polymerizations have been of particular interest owing to their potential for solar‐driven chemistry and biomedical applications. In this work, we present the first example of a novel photocatalyst for both full broadband‐ and NIR‐mediated CRP in aqueous conditions. Well‐defined polymers were synthesized in water under blue, green, red, and NIR light irradiation. Exploiting the oxygen tolerant and aqueous nature of our system, we also report PET‐RAFT polymerization at the microliter scale in a mammalian cell culture medium.

Enhancement of cell proliferation and motility of mammalian cells grown in co-culture with Pichia pastoris expressing recombinant human FGF-2

Many studies examining the biological function of recombinant proteins and their effects on the physiology of mammalian cells stipulate that the proteins be purified before being used as therapeutic agents. In this study, we explored the possibility of using unpurified recombinant proteins to treat mammalian cells. The recombinant protein was used directly from the expression source and the biological function was compared to purified commercially available, equivalent protein. The model for this purpose was recombinant FGF-2, expressed by Pichia pastoris, which was used to treat the murine fibroblast cell line, NIH/3T3. We generated a P. pastoris strain (yHL11) that constitutively secreted a biologically active recombinant FGF-2 protein containing an N-terminal c-myc epitope (Myc-FGF-2). Myc-FGF-2 was then used without purification either a) in the form of conditioned mammalian cell culture medium or b) during co-cultures of yHL11 with NIH/3T3 to induce higher proliferation and motility of NIH/3T3 cells. The effects of Myc-FGF-2 on cell physiology were comparable to commercially available FGF-2. To our knowledge, this is the first time the physiology of cultured mammalian cells had been successfully altered with a recombinant protein secreted by P. pastoris while the two species shared the same medium and culture conditions. Our data demonstrated the biological activity of unpurified recombinant FGF-2 on NIH/3T3 cells and provided a foundation for directly using unpurified recombinant proteins expressed by P. pastoris with mammalian cells, potentially as wound-healing therapeutics.

Effects of amotosalen treatment on human platelet lysate bioactivity: A proof-of-concept study.

Clinical application of mesenchymal stromal cells (MSCs) usually requires an in vitro expansion step to reach clinically relevant numbers. In vitro cell expansion necessitates supplementation of basal mammalian cell culture medium with growth factors. To avoid using supplements containing animal substances, human platelet lysates (hPL) produced from expired and pathogen inactivated platelet concentrates can be used in place of fetal bovine serum. However, globally, most transfusion units are currently not pathogen inactivated. As blood banks are the sole source of platelet concentrates for hPL production, it is important to ensure product safety and standardized production methods. In this proof-of-concept study we assessed the feasibility of producing hPL from expired platelet concentrates with pathogen inactivation applied after platelet lysis by evaluating the retention of growth factors, cytokines, and the ability to support MSC proliferation and tri-lineage differentiation. Bone marrow-derived MSCs (BM-MSCs) were expanded and differentiated using hPL derived from pathogen inactivated platelet lysates (hPL-PIPL), with pathogen inactivation by amotosalen/ultraviolet A treatment applied after lysis of expired platelets. Results were compared to those using hPL produced from conventional expired pathogen inactivated platelet concentrates (hPL-PIPC), with pathogen inactivation applied after blood donation. hPL-PIPL treatment had lower concentrations of soluble growth factors and cytokines than hPL-PIPC treatment. When used as supplementation in cell culture, BM-MSCs proliferated at a reduced rate, but more consistently, in hPL-PIPL than in hPL-PIPC. The ability to support tri-lineage differentiation was comparable between lysates. These results suggest that functional hPL can be produced from expired and untreated platelet lysates by applying pathogen inactivation after platelet lysis. When carried out post-expiration, pathogen inactivation may provide a valuable solution for further standardizing global hPL production methods, increasing the pool of starting material, and meeting future demand for animal-free supplements in human cell culturing.

Electrochemical behaviour of the MA8 Mg alloy in minimum essential medium

The difference of the protective properties of the corrosion film formed on the MA8 Mg alloy in the mammalian cell culture medium (MEM) and 0.83 wt. % NaCl solution is established using OCP, PDP and EIS tests. The impedance modulus in the frequency range from 105 Hz down to 10−1 Hz for the sample immersed in MEM is higher than that for the sample immersed in NaCl solution. Ca and P rich deposits are formed in the corrosion layer on the Mg alloy in MEM. The model of the corrosion mechanism for MA8 Mg alloy in the MEM is proposed.

Aqueous solubility of beryllium(II) at physiological pH: effects of buffer composition and counterions

Beryllium ion elicits p53-mediated cell cycle arrest in some types of human cancer cells, and it is a potent inhibitor of GSK3 kinase activity. Paradoxically, Be2+ is regarded to have almost negligible aqueous solubility at physiological pH, due to precipitation as Be(OH)2. This study demonstrates that the interaction of Be2+ with serum proteins greatly increases its effective solubility. In typical serum-supplemented mammalian cell culture medium, Be2+ was soluble up to about 0.5 mM, which greatly exceeds the concentration needed for biological activity. Some biochemical studies require protein-free Be2+ solutions. In such cases, the inclusion of a specific inorganic counterion, sulfate, increased solubility considerably. The role of sulfate as a solubility-enhancing factor became evident during preparation of buffered solutions, as the apparent solubility of Be2+ depended on whether H2SO4 or a different strong acid was used for pH adjustment. The binding behavior of Be2+ observed via isothermal titration calorimetry was affected by the inclusion of sodium sulfate. The data reflect a “Diverse Ion Effect” consistent with ion pair formation between solvated Be2+ and sulfate. These insights into the solubility behavior of Be2+ at physiological and near-physiological pH will provide guidance to assist sample preparation for biochemical studies.

Properties of a fungicidal product formed from a reaction between L-cystine and pyridoxal.

Previously we found that three components of a commonly used mammalian cell culture medium incorporated into agar killed cryptococci (Granger and Call 2019). The components were L-cystine, iron [Fe(III)], and pyridoxal (CIP). We now report on a buffered solution at neutral pH of the three components, which was highly fungicidal without agar. We showed that CIP fungicidal activity, identical to the findings with cell culture medium, was inactivated by visible light and was unstable with storage in the dark. Congeners replacing either pyridoxal or L-cystine in CIP revealed structural requirements for fungicidal activity. Replacing pyridoxal in CIP with 2-hydroxy-5-nitrobenzaldehyde produced a solution that was equally fungicidal and maintained fungicidal activity upon storage in the dark for up to 50 days. We employed methods for excluding iron from CIP and found that fungicidal activity was not affected. Upon mixing L-cystine and pyridoxal in buffer at pH 7.0, diode array spectroscopy revealed a red-shift of absorbance maximum from 391nm to 398nm. Our findings point to Schiff base reaction between the pyridoxal aldehyde group of C1 with the alpha amino group(s) of cystine to yield a fungicidal compound. Light at wave length approximately 400nm inactivates this complex accompanied by bleaching of the pyridine ring of pyridoxal. Our findings may be useful for design of a class of fungicidal compounds formed through Schiff base reaction of disulfide compounds with aromatic ring-bearing aldehydes.

During the Early Stages of Staphylococcus aureus Biofilm Formation, Induced Neutrophil Extracellular Traps Are Degraded by Autologous Thermonuclease.

Staphylococcus aureus extracellular DNA (eDNA) plays a crucial role in the structural stability of biofilms during bacterial colonization; on the contrary, host immune responses can be induced by bacterial eDNA. Previously, we observed production of S. aureus thermonuclease during the early stages of biofilm formation in a mammalian cell culture medium. Using a fluorescence resonance energy transfer (FRET)-based assay, we detected thermonuclease activity of S. aureus biofilms grown in Iscove's modified Dulbecco's medium (IMDM) earlier than that of widely studied biofilms grown in tryptic soy broth (TSB). The thermonuclease found was Nuc1, confirmed by mass spectrometry and competitive Luminex assay. These results indicate that biofilm development in IMDM may not rely on eDNA for structural stability. A bacterial viability assay in combination with wheat germ agglutinin (WGA) staining confirmed the accumulation of dead cells and eDNA in biofilms grown in TSB. However, in biofilms grown in IMDM, minimal amounts of eDNA were found; instead, polysaccharide intercellular adhesin (PIA) was detected. To investigate if this early production of thermonuclease plays a role in immune modulation by biofilm, we studied the effect of thermonuclease on human neutrophil extracellular trap (NET) formation using a nuc knockout and complemented strain. We confirmed that thermonuclease produced by early-stage biofilms grown in IMDM degraded biofilm-induced NETs. Additionally, neither the presence of biofilms nor thermonuclease stimulated an increase in reactive oxygen species (ROS) production by neutrophils. Our findings indicated that S. aureus, during the early stages of biofilm formation, actively evades the host immune responses by producing thermonuclease.

Regulatory effects of PRF and titanium surfaces on cellular adhesion, spread, and cytokine expressions of gingival keratinocytes.

Dental implant material has an impact on adhesion and spreading of oral mucosal cells on its surface. Platelet-rich fibrin (PRF), a second-generation platelet concentrate, can enhance cell proliferation and adhesion. The aim was to examine the regulatory effects of PRF and titanium surfaces on cellular adhesion, spread, and cytokine expressions of gingival keratinocytes. Human gingival keratinocytes were cultured on titanium grade 4, titanium grade 5 (Ti5), and HA discs at 37°C in a CO2 incubator for 6h and 24h, using either elutes of titanium-PRF (T-PRF) or leukocyte and platelet-rich fibrin (L-PRF), or mammalian cell culture medium as growth media. Cell numbers were determined using a Cell Titer 96 assay. Interleukin (IL)-1β, IL-1Ra, IL-8, monocyte chemoattractant protein (MCP)-1, and vascular endothelial growth factor (VEGF) expression levels were measured using the Luminex® xMAP™ technique, and cell adhesion and spread by scanning electron microscopy. Epithelial cell adhesion and spread was most prominent to Ti5 surfaces. L-PRF stimulated cell adhesion to HA surface. Both T-PRF and L-PRF activated the expressions of IL-1 β, IL-8, IL-1Ra, MCP-1, and VEGF, T-PRF being the strongest activator. Titanium surface type has a regulatory role in epithelial cell adhesion and spread, while PRF type determines the cytokine response.